Rhodococcus equi is an encapsulated and rod shaped, Gram positive bacterium that is considered to be a soil saprophyte that survives well in the environment. R. equi has long been considered a pathogen in horses principally in foals fewer than 6 months old (particularly 1–3 months old). Infection by the organism is accompanied by extra-pulmonary manifestations, causes a pyogranulomatous pneumonia, often such as bacteraemia, lymphadenitis, meningitis and enteritis (Barton and Hughes, 1980; Giguere and Prescott, 1997; Takai, 1997). Infections are often fatal if untreated. Apart from causing disease in horses, R. equi also causes infections in cattle, pigs and goats (Barton, 1992). R. equi is also known to cause severe pulmonary and disseminated disease in immuno-compromised humans, particularly AIDS patients (Capdevila et al., 1997).
In Australia most equine R. equi infections occur in summer (December to February) when the age of the foals as well as the warm and dry environmental conditions make the animals more susceptible to infection (Barton and Hughes, 1984).
R. equi produces a range of putative virulence factors such as cholesterol oxidase, phospholipase C and lecithinase (Smola et al. 1994). However one of the more important putative virulence factors is considered to be a 17 kDa virulence associated protein (VapA) which is plasmid encoded. This protein is known to be produced by up to 90% of equine clinical isolates of R. equi. Although VapA producing strains are widespread among disease causing isolates, recent work has shown that VapA protein alone is not sufficient to cause disease in foals and that other as yet unknown plasmid borne factors are likely to be involved (Giguere et al., 1999). The role of VapA in virulence is yet to be elucidated, although there is strong evidence to suggest that the plasmid encoding the protein may play an important part in the survival of the organism within macrophages (Hondalus and Mosser, 1994).
Current techniques for the identification of R. equi such as culture and phenotypic testing are often not successful due to the slow growing nature of the organism. It is also often difficult to differentiate R. equi from closely related organisms based on biochemical tests alone. It has been shown that the detection of high levels of IgG antibodies to the VapA protein is an indicator of R. equi infection in horses (Prescott et al., 1996; Higuchi et al., 1997). Of the immunodiagnostic tests developed to date, none have been found suitable for use on a routine basis mainly due to the use of laborious protein extraction techniques (Higuchi et al., 1997; Takai, 1997). Thus an improved immunodiagnostic test based on the VapA protein would be of great benefit as a routine diagnostic test.
Further, attempts at immunisation against Rhodococcus infection have met with limited success to date. The use of antibiotics has been found to be only partially effective and vaccines developed over the years for the prevention of R. equi infection in horses have not been particularly effective.
Mosser in WO 99/05304 has found that an avirulent strain transformed with a VapA expressing plasmid is avirulent but is also protective and describes a vaccine comprising DNA encoding the VapA protein or fragment thereof. Mosser's results are based on the immunogenicity of the whole VapA protein. However, Mosser has found that VapA is poorly immunogenic in mice (personal communication).
Peptides, and particularly relatively small peptides, have an advantage over whole proteins in diagnostics and therapeutics in that they are more readily produced than the whole protein, and they generate a population of homogenous molecules, i.e. single peptides composed of the same amino acids. Further it may not be economically viable to synthesise large proteins and therefore native proteins are often obtained by extraction. However native proteins derived from natural sources may contain other proteins or peptides of the same origin as the target proteins and as such the complexity and variability of mixtures of native antigen proteins can persist even after fractionation and purification and this can be a barrier to their use in immunoassays and vaccines.
For these reasons it may be more preferable if a vaccine were based on one or more small peptides that have the immunogenic properties of the whole VapA protein. However, Mosser does not identify any peptide fragments that provide for recognition of antibodies specific for VapA. Further, it is often difficult to identify peptide fragments that mimic immunogenic properties specific for a whole protein because in many cases the peptide fragments do not take up the three dimensional structure necessary for immunological recognition.